Figure 6.

Overexpression of Ste9 prevents mitosis and induces rereplication of the genome. (A) Changes in the Cdc13 level and the H1-histone kinase activity during Ste9-overexpression. Cells harboring the integrated nmt-ste9⁺ gene were cultured to mid-log phase in the presence of thiamine. At 0 h, the cells were washed and then reinoculated into fresh medium without thiamine (ON) or with thiamine (OFF). Cell extracts were prepared from the cultures at the times indicated. Note that induction from the nmt promoter requires more than 10 h (Maundrell, 1990). Immunoblotting was carried out by anti-Cdc13 antibody or by anti-PSTAIRE antibody (upper two panels). Arrow and asterisk indicate the locations of Cdc13 and cross-reacted proteins, respectively. H1-histone kinase activities of uninduced (OFF) and induced (ON) cultures were also measured (lower panel). (B) Overexpression of Ste9 inhibits cell division. Cell numbers of induced (ON) and uninduced (OFF) cultures were counted at the indicated time. (C) Changes in septation index in the same cultures described in panel B. (D) Changes in the DNA content during Ste9 overexpression. Samples were taken at the indicated time for flow cytometry. The DNA profiles of the uninduced culture at each time were the same as that of 0 h. (E) The ste9 mutation prevents rereplication of DNA due to heat inactivation of the temperature-sensitive Cdc2 protein. Log-phase cells were shifted to EMM2−N and cultured at 36°C for 6 h. Cells were plated at 0 h (open bar) and at 6 h (black bar) onto YE medium containing phloxine B. Numbers of red colonies (diploid cells) and pink colonies (haploid cells) were counted, and the percent of diploid cells was calculated. Strains: cdc2-M26 (KJ141–8A); Δste9 cdc2-M26 (KJ124–3A); Δchk1 cdc2-M26 (KJ155–1A).